Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

doi: 10.1073/pnas.2321167121

Figure Lengend Snippet: Fig. 6. Crystal structure of the human CTDNEP1–NEP1R1 phosphatase complex. (A) Domain architecture of the crystallized CTDNEP1ΔAH–sNEP1R1 fusion with linker sequence. (B) Quantification of pNPP hydrolysis by CTDNEP1ΔAH /sNEP1R1 copurified complex and the CTDNEP1ΔAH–sNEP1R1 fusion. Error bars represent SD (n = 3). (C) Overall structure of the CTDNEP1–NEP1R1 phosphatase complex. A linker peptide occupies the active site of the CTDNEP1 catalytic subunit. NEP1R1 forms an extended helix that packs against the opposite side of the CTDNEP1 active site. (D and E) Interactions between NEP1R1 and CTDNEP1 (D) observed in the crystal structure and (E) predicted by AlphaFold multimer. (F) Size-exclusion profiles (solid lines) and quantification pNPP activity (dotted lines) of MBP- CTDNEP1ΔAHS232D (blue traces), NEP1R1 (red traces), and a mixture of CTDNEP1ΔAHS232D+NEP1R1 (purple traces). (G) Quantification of pNPP hydrolysis by MBP-CTDNEP1ΔAH WT and S232D with and without sNEP1R1. Error bars represent SD (n = 3). (H) Melting temperatures (Tm’s) of the His-sNEP1R1/CTDNEP1ΔAH complex in comparison to WT, S232D, and V233E MBP-CTDNEP1ΔAH with and without sNEP1R1. sNEP1R1 increases the stability of MBP-CTDNEP1ΔAH but not the point mutants S232D and V233E that disrupt the complex. P-values were determined by an unpaired t test. (n = 2, or n = 1 for MBP alone).

Article Snippet: 4- Nitrophenyl phosphate (pNPP), NickelNTA Agarose resin, and n- Dodecyl- β- D- Maltopyranoside (DDM) were from Gold Biotechnology. n- Decyl- β- D- Maltopyranoside (DM), and glyco- diosgenin (GDN) were products of Anatrace.

Techniques: Sequencing, Activity Assay, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

doi: 10.1073/pnas.2321167121

Figure Lengend Snippet: Fig. 7. Peptide recognition by CTDNEP1. (A) Model of the linker peptide bound in the CTDNEP1 active site with the 2Fo-2Fc electron density map contoured at 1σ. (B) CTDNEP1 residues involved in binding the linker peptide. Intermolecular hydrogen bonds between the conserved Arginine residue and the carbonyl groups of the linker peptide are shown as black dotted lines. (C) Scp1 residues involved in binding a phospho-peptide. Intermolecular hydrogen bonds between the conserved Arginine residue and the carbonyl groups of the phosphor-peptide are shown as black dotted lines. (D) Superimposition of the CTDNEP1 and Scp1 structures with bound peptides. (E) Quantification of pNPP hydrolysis by MBP-CTDNEP1ΔAH WT, E70S, and R158A. Error bars represent SD (n = 3). (F) Phos-tag SDS-PAGE analysis of dephosphorylation of mouse lipin 1α after treatment with purified MBP-CTDNEP1ΔAH WT, E70S, and R158A proteins.

Article Snippet: 4- Nitrophenyl phosphate (pNPP), NickelNTA Agarose resin, and n- Dodecyl- β- D- Maltopyranoside (DDM) were from Gold Biotechnology. n- Decyl- β- D- Maltopyranoside (DM), and glyco- diosgenin (GDN) were products of Anatrace.

Techniques: Binding Assay, Residue, SDS Page, De-Phosphorylation Assay, Purification